Variation in thickness and protein content of the cuticle of the female of Glossina austeni.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 4:287-296 (1987) Variation in Thickness and Protein Content of the Cuticle of the Female of Clossina austeni S. Solowiej and K.G. Davey Department of Biology, York University, Downsview, Ontario, Canada The thickness and total protein content of the ventral abdominal cuticle of the female tsetse, Glossina austeni, increase during the early part of each pregnancy cycle, reaching a maximum at approximately 2 days after ovulation. They decrease thereafter, and reach a minimum value just before larviposition. Virgin females do not exhibit a cycle of protein content or thickness in the cuticle. Preliminary data on the incorporation of j3H]tyrosine or [3H]leucine into the water-soluble proteins of the ventral abdominal cuticle at the time of the second larviposition suggest that there i s rapid turnover of protein i n the cuticle at this time. These observations are consistent with the net storage of protein in the cuticle during the early part of pregnancy cycle followed by a net depletion of that store as the nutritional demands of the rapidly growing larva in utero exceed the capacity of the ingested blood meals to supply them. Key words: protein synthesis, tsetse, insect nutrition INTRODUCTION Tsetse are hematophagous and reproduce by adenotrophic viviparity. The female tsetse imbibes a series of blood meals which are used to nourish the larva, which develops within the uterus of the female, via the secretion of the milk gland of the mother. In the laboratory colony of Glossina atrsteni used in this study, the first ovulation occurs on the ninth or tenth day, and larvae are deposited every 9 days thereafter. The mated female feeds to a constant weight, and the meals later in the cycle of pregnancy, when the nutritional demands of the large and growing larva are maximal, are considerably smaller than those taken earlier in the cycle [l]. These facts imply that nutrients from the larger earlier blood meals may be stored against the requirements later in the cycle. In G. rnorsitans, it has been suggested that Acknowledgments: Research in this laboratory is supported by grants from the Natural Sciences and Engineering Research Council of Canada. Received June 23,1986; accepted September 25,1986. Address reprint requests to K.G. Davey, Department of Biology, York University, Downsview, Ontario M3J1P3 Canada. 0 1987 Alan R. Liss, Inc. 288 Solowiej and Davey the increased secretion of milk during the latter half of pregnancy may involve the mobilisation of stored nutrients from tissues other than the fat body or milk gland . In a study of the elastic properties of the abdominal cuticle of G. austeni, it became apparent that the content of water-soluble protein in the cuticle varied during the pregnancy cycle . This raised the possibility that the cuticle of this species might act as a store for protein, which might accumulate in the cuticle early in the cycle to be released later in pregnancy. The present paper represents a preliminary approach to this hypothesis. It investigates changes in the thickness and in total protein content of the cuticle during the pregnancy cycle. It also presents preliminary data on the rate of incorporation of tritiated leucine and tyrosine into water soluble proteins of the cuticle at the end of the pregnancy cycle. Comparisons with virgin females of similar age provide controls for these measurements. MATERIALS AND METHODS The insects were maintained at 25°C and fed on alternate days on the ears of rabbits, as described by Tobe and Davey . Experimental females were isolated at emergence from the puparium and maintained singly; some of these females were mated on the third day of adult life, while others remained unmated. The flies were allowed to feed on alternate days. Scanning Electron Microscopy Whole abdomens were excised, fixed for 2 h in cold 5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4), rinsed in distilled water, and dehydrated in ethanol. Following critical point drying, the abdomens were cut transversely across the third segment, taking great care to make the cut perpendicular to the longitudinal axis of the abdomen. The abdomens were mounted on specimen stubs, coated with gold-palladium, and viewed in a Hitachi S520 scanning electron microscope. In making measurements of the thickness of the cuticle in the scanning electron microscope, it is essential that the cut surface of the cuticle be perpendicular to the electron beam. When that condition is met, both the outer and inner edges of the cuticle will be in the same focal plane. The specimen was viewed at a magnification of ~12,000, with the goniometer stage adjusted until both edges were in focus at the same focal setting. The specimens were photographed, and measurements of the thickness taken from the photographs. The measurements were made on alternate days, 24 h after feeding, throughout the first two cycles of pregnancy. Total Protein and Chitin The entire dorsal and ventral cuticles of the abdomen were separated and carefully cleaned of epidermal cells and adhering tissue. The cuticles were individually dried at 100°C to a constant weight as determined on a microbalance. The cuticles were heated at 110°C in sealed tubes with 1 ml 2.5 N NaOH for 2 h, washed successively in water, 1N HCl ethanol, and diethyl Protein in Cuticle of Tsetse 289 ether. The remaining material, assumed to be chitin , was dried to a constant weight. Total protein was the difference between the dry weight of the cuticle and the dry weight of the chitin. These determinations were made on alternate days, 24 h after feeding, throughout the first two cycles of pregnancy. Incorporation of [3H]Leucine and [3H]Tyrosine The mated females in this experiment were in their second cycle of pregnancy, thus permitting the precise timing of the measurements relative to ovulation, which coincides with the first larviposition. Virgin females were of equivalent chronological age. They were injected in the thorax with 1p1 of 2% ethanol containing 1pCi of L-[3,5-3H]leucine(Amersham-Searle, Toronto, Canada, 55.5 mCilmmo1) or of L-[3,5-3H]tyrosine (Amersham-Searle, 51.0 mCilmmo1). All injections were performed approximately 6-8 h prior to larviposition. Incorporation of the amino acids into water-soluble proteins was determined by the methods of Mans and Novelli . In brief, the ventral cuticle was dissected out, cleaned of epidermis and other tissue, soaked in 50 pl of distilled water, homogenised, and the suspension centrifuged at 10,000 g for 10 min. Aliquots (25 pl) were spotted onto filter paper and processed according to Mans and Novelli  before transferring the papers to scintillation fluid (3.0 g 2,5 diphenyloxazole, 0.3 g 1,4 bis[2-(phenyloxaznlyl)]-benzene in 1liter toluene). After counting, the protein on the filter papers was determined by the method of Bramhall et al , with bovine serum albumin as a standard . RESULTS Cuticle Thickness Figure 1 shows the thickness of the ventral abdominal cuticle of mated females throughout the first two cycles and of virgin females of the same age. In mated females, there is an increase in the thickness of the cuticle from the fifth day of adult life (about 2-3 days before the first ovulation) until the eleventh day, about midway through the cycle of pregnancy. Thereafter, the thickness decreases sharply up to the time of larviposition and ovulation, when the cuticle increases sharply once more, reaching a peak 2 days after ovulation. During the later part of the pregnancy cycle, the thickness of the cuticle declines once more. By contrast, the thickness of the cuticle from virgin females does not exhibit the same cyclical variation: it increases during the first 9 days of adult life, but thereafter does not vary appreciably. Total Protein Content Figures 2 and 3 display the total protein and the chitin content of the dorsal and ventral abdominal cuticles for virgin and mated females at various times during adult life. The total protein content of the ventral abdominal cuticle undergoes a cycle which closely resembles the changes in thickness, 290 Solowiej and Davey let Age (days) Fig. 1. The thickness of the ventral abdominal cuticle of mated (0-0) and virgin (0-- - -0)females of G. austeni as determined at various times after emergence. L1 and L2 indicate the times of the first and second larvipositions. The lines have been drawn through the means of from 5-9 determinations, and the vertical lines indicate the standard deviations of the means. r T 280. A 240- 200. 160- ..0 120- P 80. m -.. r 4 8 12 16 20 24 Age (days) Fig. 2. The total protein content (A) and chitin content (6) of the ventral abdominal cuticle of mated (0-0) and virgin (0-- - -0) females of G. austeni at various times after emergence. L1 and L2 indicate the times of the first and second larvipositions. The lines have been drawn through the means of from 9-11 determinations, and the vertical lines indicate the standard deviations of the means. Protein in Cuticle of Tsetse 291 A T 240. -* - 200- k i60- + . : 120- - li : 80. t I 0 B . . 4 * . 8 . . 12 . . 16 . 20 . . 24. Age (days) Fig. 3. As in Figure 2, but for dorsal cuticles. although the peak of protein content during the first cycle occurs somewhat earlier than the peak of thickness. The ventral cuticles from virgin females exhibit an increase during the early part of adult life, but do not exhibit a cycle in protein content. The protein content of the dorsal cuticle behaves in a similar fashion, although the amplitude of the cycle exhibited is not so great, particularly in the first cycle. These data should be viewed against the background of possible changes in the content of chitin in the cuticle, since the protein contents are derived from determinations of chitin. As can be seen from Figures 2 and 3, the chitin content of the cuticle increases during the first 11 days of adult life, but remains more or less constant thereafter. Thus, it is clear that the total protein content of the cuticle of mated females varies independently of the chitin content during the pregnancy cycle. Incorporation of Amino Acids Table 1displays the incorporation of [3H]tyrosineinto water-soluble cuticular proteins of mated and virgin females at various times after a single injection. In virgin females, label accumulated in the water-soluble proteins of the cuticle up to at least 24 h after injection of the precursor. By 42 h, however, the amount of label had decreased sharply, and by 48 h it was no longer detectable. During this time there was no appreciable change in the amount of water-soluble protein which could be extracted from the cuticle. 292 Solowiej and Davey TABLE 1. Incorporation of [3H]Tyrosine Into Water-Soluble Protein of the Ventral Abdominal Cuticle of Mated and Virgin Females of G . austeini at Various Times After Injection of 1 pCi of the Isotope? p g protein/ Time (h) Mated 1 2 4 12* 18 24 36 45 N dpmi cuticle cuticle 5 5 5 4 5 5 5 3 73.5 f 83.5 121.8 f 108.0 142.4 f 98.2 183.6 f 94.5 215.8 f 56.8 392.7 k 189.5 106.7 k 133.4 0.6 k 6.0 33.4 k 12.2 39.3 k 10.7 49.1 f 8.8 54.0 f 9.3 56.8 5 9.5 68.9 f 7.2 48.5 5 10.9 30.2 k 8.7 2.2 3.1 2.9 3.4 3.8 5.7 2.2 0.2 5 5 4 5 5 5 4 4 7,637.9 1,165.5 7,994.4 f 1,565.9 11,159.9 f 1,591.9 15,953.8 _+ 1,682.0 16,992.0 f 1,200.0 26,847.2 f 1,480.0 6,058.5 f 1,527.9 0.0 f 77.7 5 11.3 78.3 f 8.7 79.6 _+ 6.4 84.1 f 7.9 80.0 5 7.7 74.9 f 9.2 76.4 f 7.1 78.3 f 8.2 98.3 102.1 140.2 189.7 212.4 362.8 79.3 0.0 Virgin 1 2 6 12 18 24 42 48 * SPA ?.SPA:specific activity in dpmipg protein. The values are given as means k SD. * Indicates the time of the second lamiposition. In mated females of G. austeni, the amount of water-soluble protein increased over the first 24 h of the period after injection, and then decreased. Both the amount of label and the specific activity of the water-soluble proteins increased and then decreased in the same way as in the cuticle from virgin females. However, the amount of label incorporated was very much less in the mated females. Table 2 displays similar data related to the incorporation of [3H]leucine into water-soluble proteins of the cuticle. In virgin flies, the amount of label incorporated increases over the first 18 h after injection, but begins to decrease by 24 h after injection. During the period of observation, the amount of water-soluble protein in the cuticle did not vary in an appreciable way. As with the experiments involving tyrosine, the amount of water-soluble protein in the cuticles from mated females increased up to 24 h after injection and then decreased. Both the amount of [3H]leucine incorporated and the specific activity of the water-soluble protein exhibited a similar cycle. Once more the amount of label incorporated into the cuticular proteins of virgin females was markedly greater than in mated females. DISCUSSION The facts presented in this paper are consistent with the view that the cuticle in mated female G. austeni acts as a storage organ for protein. Thus, there is an increase in thickness of the cuticle early in the pregnancy cycle, followed by a decrease in the latter half of the cycle, when the demands of Protein in Cuticle of Tsetse 293 TABLE 2. Incorporation of t3H] Leucine Into Water-Soluble Protein of the Ventral Abdominal Cuticle of Mated and Virgin Females of G . austeni at Various Times After Injection of 1 pCi of the Isotope? Time (h) Mated 1 2 6 12* 18 24 35 42 Virgin 1 2 6 12 18 24 36 48 N dpmlcuticle p g proteinlcuticle SpA 5 5 5 5 4 5 4 5 16.5 f 5.4 23.3 f 5.5 39.5 f 13.9 54.2 rt 10.7 74.7 f 8.7 14.4 f 13.1 5.8 f 9.2 8.4 f 5.5 31.2 f 9.0 36.4 f 9.6 47.0 f 10.4 53.7 f 7.8 57.9 f 9.3 65.6 f 8.9 52.3 f 11.2 36.4 f 7.5 0.53 0.64 0.84 1.01 1.29 0.22 0.11 0.23 5 5 5 5 5 4 5 4 110.7 f 14.8 111.4 f 10.8 122.9 f 9.7 159.3 f 22.9 252.1 f 22.4 174.7 f 20.0 134.5 f 18.5 76.8 f 11.4 73.8 f 7.0 71.9 f 7.0 77.3 f 7.3 76.6 f 10.7 74.8 f 7.6 72.8 f 9.2 73.9 f 8.3 76.1 f 7.9 1.50 1.55 1.59 2.08 3.37 2.40 1.82 1.01 ?The values are given as means rt SD. * Indicates the time of the second larviposition. the rapidly growing larva are large and the meals are decreasing in size. Moreover, this increase and decrease in thickness is reflected by a similar and marked increase and decrease in the non-chitinous, and by inference, proteinaceous  moiety of the cuticle, while the content of chitin remains approximately constant during the pregnancy cycle. The view that this cycle of protein content is associated with pregnancy is strengthened by the observation that the cycle is absent from virgin females of similar age. The variation in thickness and protein content of the cuticle is not a direct consequence of alterations in the stretch of the cuticle during the pregnancy cycle. It is well established for G. austeni that pregnant females feed to a constant volume [l], so that the maximum degree of stretch during a feeding cycle is constant throughout the pregnancy cycle. When a female feeds, the increase in volume occurs almost exclusively by stretching of the ventral abdominal cuticle. While the magnitude of the variation in protein content is very much less marked, it is evident that the dorsal cuticle also participates in the cycle. These data confirm and extend those provided by Carruthers , who found that both the elasticity and the content of water soluble protein of the ventral cuticle of G. austeni increased over the first 11days of adult life, and that the content of water-soluble protein was higher in the cuticles of virgin females, which are not stretched as much, than in those of mated females. Moreover, her studies revealed that there was no clear relationship between the content of water-soluble protein and the elasticity of the ventral cuticle. In order to investigate further the possibility that nutrients were being incorporated into the cuticle, the incorporation of leucine and tyrosine into 294 Solowiej and Davey the water-soluble proteins of the ventral abdominal cuticles was investigated at about the time of larviposition. This particular time was selected as one in which the shift from a net depletion mode to a net storage mode was occurring. The decision to examine only the water-soluble proteins was based on the assumption that this part of the cuticle would be most likely to exhibit short-term fluctuations. The resulting data lead to several provocative conclusions. First, there is a difference in the pattern of incorporation between t rosine and leucine. In both virgin and mated females, the incorporation of [ Hlleucine reached its peak about 18 h after injection, and by 24 h the incorporation had begun to decline. For r3H]tyrosine, however, the incorporation was still increasing at 24 h postinjection. This difference, and the pattern of incorporation of the amino acids into the proteins of the cuticle, must be viewed against a complex background of the utilisation of amino acids in tsetse. It is clear that the labelled pool of amino acids in G. austeni is depleted within 1h of injection, and that incorporated amino acids are later released into the hemolymph as trichloroacetic acid-soluble material [9, 101. This movement of amino acids into and out of other tissues explains the fact that incorporation into cuticular proteins continues beyond the time that the radioactive pool is depleted. In addition, however, there is also evidence that tyrosine may undergo some additional processing, perhaps related to increasing its solubility, which prolongs the availability of labelled precursors for incorporation into proteins [lo]. Injected amino acids will be subjected to intense competition among the fat body, ovary, and uterine gland for incorporation into protein: it is thus scarcely surprising that of the 200,000 dpm injected, relatively trivial amounts find their way into the ventral cuticle. Similarly, it is no surprise to find that the rates of incorporation into the cuticle of virgin females is very much higher than those into the cuticles of mated females. In virgins, because there is no larva, the milk gland is relatively inactive, and the competition from the prominent structure and other tissues will be less intense. Secondly, and perhaps most importantly, there are clear signs of rapid turnover of cuticular proteins. In virgin females, in which the total content of water-soluble cuticular protein does not change very much during the period after injection, the label first accumulates and then disappears. Moreover, in mated females, the incorporation of [3H]leucine decreases at a time when the content of water-soluble protein is still increasing. Taken together, these facts render it unlikely that the pattern of incorporation of labelled amino acids and content of water-soluble protein is wholly the result of the conversion of water-soluble protein into insoluble cuticular protein. Indeed, at the time surrounding parturitionlovulation, there is no net conversion of water-soluble protein into insoluble cuticular protein. Given that at the time of the injection the water-soluble protein was increasing while the results from the experiments on total protein suggest that the total protein may have been decreasing, there may have been a net conversion from insoluble to soluble. In any case, it is clear that at least the water-soluble proteins of the cuticle of the female of G. austeni are in a highly dynamic state. Thirdly, in mated females, the amount of water-soluble protein increases over the period from about 12 h before larviposition until about 12 h after, Y Protein in Cuticle of Tsetse 295 when it begins to decline. There are many possible explanations for such an event, but the most likely involves the increasing demands of the milk gland. Thus, as the end of the cycle approaches, the synthetic activity of the milk gland wanes, while the material in its lumen continues to be transferred to the larva, resulting in a decrease in diameter of the gland . This reduction in synthetic activity makes more metabolites available for incorporation into cuticular protein. At parturitionlovulation, the milk gland becomes synthetically active, and begins to store secretion against the needs of the new larva ; this increases the competition for the available amino acids, and the amount of cuticular material begins to decrease. It is appropriate to consider whether the protein available in the watersoluble pool in the cuticle is significant in the overall nutrition of the tsetse. From the data presented here, about 250 pg of protein (dry weight) leaves the dorsal and ventral abdominal cuticles during the second cycle of pregnancy. Given that the wet weight of a mature larva is about 30 mg, and assuming a water content of approximately yo%, then the dry weight of a larva is about 9 mg. The protein stored in, and released from, the abdominal cuticle represents approximately 3% of the weight of the larva. It has been determined [ll] that a reduction of only 10% in the food intake of the female of G. rnorsitans results in a reduction of at least 20% in the weight of the pupa produced. Since larger pupae exhibit increased longevity as adults [El, there is good reason to conclude that the temporary storage of protein in the cuticle may be an important element in the nutritional strategy of the tsetse. The situation in G. austeni resembles that in Rhodnius, in which the endocuticle becomes reduced and thin after some months of starvation, losing as much as 40% of the cuticular dry weight . However, the rapidity of the turnover of the labelled amino acids in G. austeni suggests that the watersoluble protein in the cuticle represents a highly dynamic storage site for the protein. Whether the changes in the content of water-soluble protein are the result of changes in the rate of deposition of the protein or of its removal remains to be seen. In addition, the question of some central control over the content of the protein needs investigation. LITERATURE CITED 1. Tobe SS, Davey KG: Volume relationships during the pregnancy cycle of the tsetse fly Glossina austeni. Can J Zool 50, 999 (1972). 2. Langley PA, Pimley RW: (1975) Quantitative aspects of reproduction and larval nutrition in Glossina rnorsitans rnorsitans West. fed in vitro. Bull Entomol Res 65, 129 (1975). 3. Carruthers CB, Davey KG: Does cuticular elasticity regulate the size of the blood meal imbibed by female Glossina austeni. Can J Zool 61, 1888 (1983). 4. Tobe SS, Davey KG: Some modifications for the laboratory rearing of GIossina austeni Newst. Can J Zool 49, 577 (1971). 5. Hackman RH, Goldberg M: Studies on the hardening and darkening of insect cuticles. J Insect Physiol 17, 335 (1971). 6. Mans RJ, Novelli GD: Measurement of the incorporation of radioactive amino acids into protein by a filter paper disk method. Arch Biochem Biophys 94, 49 (1961). 7. Bramhall S, Noack N, Wu M, Loewenberg JR: A simple colorimetric method for determination of protein. Anal Biochem 31, 146 (1969). 8. Carruthers CB: Factors Affecting the Elasticity of the Abdominal Cuticle in the Female Tsetse Fly, Glossina austeni. M. Sc. Thesis, York University, 130 pp (1982). 296 Solowiej and Davey 9. Tobe SS, Davey KG: Autoradiographic study of protein synthesis in abdominal tissues of Glossina austeni. Tissue Cell 6, 255 (1974). 10. Tobe SS, Davey KG: Synthesis and turnover of haemolymph proteins during the reproductive cycle of Glossina austeni. Can J Zoo1 53, 614 (1975). 11. Langley PA, Bursell E: Role of the fat body and uterine gland in milk synthesis by adult female Glossina rnarsitans. Insect Biochem 20, 11 (1980). 12. Leegwater-van der Linden ME: Effect of a four-day-a-week feeding regimen versus daily feeding on the reproduction of Glossina pallidipes. Ent Exp Appl29, 169 (1981). 13. Hillerton JE: Changes in the structure and composition of the extensible cuticle of Rhodnius prolixus through the fifth larval instar. J Insect Physiol24, 399 (1978).